Epstein-Barr virus (EBV) is a human herpesvirus which establishes life- long infections and is associated with several malignancies, including Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma. A central feature of the life cycle and pathogenesis of EBV is a latent state of infection in which the viral genome is maintained as an autonomously replicated plasmid and in which a restricted number of viral genes, in some cases only the gene encoding the nuclear protein EBNA1, is expressed. EBNA1 has been presumed to perform an essential role in maintaining circular EBV genomes in latently infected cells through its interactions with the EBV replication origin, oriP. A detailed understanding of how EBNA1 functions could be key to learning eventually how to eliminate latent infections by EBV. Previous studies using recombinant plasmids revealed that one element of oriP serves as an origin of DNA replication while another element serves to activate the origin but has additional activities as well, performing a DNA maintenance function that is distinct from replication and serving as a transcriptional enhancer. All of these activities are mediated by binding of EBNA1 at multiple sites. Until recently it has not been possible to test the significance of these activities to maintenance of the viral genome itself, and some recent findings suggest that the origin function of oriP might not be essential. Genetic studies of EBV will be designed to test the importance of EBNA1 and of each element of oriP for replication and maintenance of EBV genomes during latent infection and for immortalization of B cells. To help to unravel how EBNA1 performs its specific functions of DNA replication, DNA maintenance, and enhancer activation, extensive random mutagenesis of the EBNA1 gene will be used to obtain mutations that will either separate the functions or identify relationships among them. The cellular factors with which EBNA1 must interact to exert its functions will be sought using affinity methods in vitro and screening cDNA expression libraries in yeast.